The hyaluronic acid is an important polysaccharide, composed of two repeating units of β-(1,3)-D-glucuronic acid and β-(1,4)-N-acetyl-D-glucosamine. The molecular weight, depending on the method of isolation and on the source material, is within the range from 5·104 to 5·106 g·mol−1. The hyaluronic acid or its sodium salt hyaluronan is an essential component of connective tissues, synovial fluid of joints, and plays an important role in biological processes, such as hydratation, organization of proteoglycans, cell differentiation, proliferation and angiogenesis. The hyaluronic acid is a considerably hydrophilic polysaccharide and is soluble in water in the form of a salt in the whole range of pH.

Oxidation of the Hyaluronic Acid
Oxidation of polysaccharides is a process in which the oxidation degree of the polysaccharide functional groups is changing. Most frequently, carboxylic acids or aldehydes are formed, which can dramatically change the properties of the polysaccharide. In most cases, the reaction is performed by use of agents containing atoms in higher oxidation degrees.
The method of selective oxidation of saccharides on the primary hydroxyl group, described in Angelino, European Journal of Organic Chemistry 2006, 19, 4323-4326, the system of 2,2,6,6-tetramethyl-1-piperidinyloxyl radical TEMPO/TCC in DMF at the temperature of 0° C. was used, with the corresponding aldehyde as the main product.
TEMPO is a relatively stable radical which can exist in reaction in three redox forms. Only the oxidized form of TEMPO is effective for alcohols or geminal diols oxidation.

A catalytic amount of TEMPO is added to the reaction and therefore, it is necessary to use a co-oxidant which restores the presence of the TEMPO oxidized form.
2,2,6,6-tetramethyl-1-piperidinyloxyl radical (TEMPO)- and NaOCl-mediated oxidation of the primary hydroxyl group of hyaluronan to a carboxylic acid was performed at pH 10.2 and at the temperature of 0° C. (Scheme 2) (Carbohydr Res 2000, 327 (4), 455-61).
Analogous to other polysaccharides, a high regioselectivity and slight degradation of the polymer were observed. An increase of the concentration of the salt (NaBr, NaCl, Na2SO4) in the solution caused a decrease in the oxidation rate.
Oxidation of hyaluronan by use of TEMPO/NaClO system was described in the patent application WO 02/18448 A2. The authors also dealt with interactions of percarboxylated polysaccharides, while forming biological complexes.
The rate of oxidation of HA and other polysaccharides by use of sodium periodate was studied by Scott et al. (Scheme 3) (Histochemie 1969, 19 (2), 155-61).
The factors such as the chain length, substitution, polymer configuration and temperature were studied and quantified. The use of NaIO4 for an oxidation of hyaluronan was also disclosed in the U.S. Pat. No. 6,683,064 and U.S. Pat. No. 6,953,784.
Model reactions of HA analogues with low molecular weight in a physiological buffer were studied (Carbohydr Res 1999, 321, (3-4), 228-34). Oxidation products of the glucuronic and glucosamine parts were identified by GC-MS analysis. The results also suggest that the oxidation is performed primarily on the glucuronic part, while the meso-tartaric acid is the main product and may be used as a biomarker of the hyaluronan oxidation.
Use of an Oxidized HA in Cross-Linking Reactions
The use of an oxidized HA for the preparation of cross-linked hydrogels was described by Weng et al. (Scheme 4), J Biomed Mater Res A 2008, 85 (2), 352-65. Two precursors were used in this case: a partially oxidized hyaluronan and gelatin:
The physico-chemical properties of the resulting hydrogels have been elucidated by instrumental analyses FT-IR, SEM (scanning electron Microscopy) and rheometry. Increasing the oxidation degree of the hyaluronan lead to a corresponding increase of hydrogels compatibility and decrease of water absorption capacity. Dermal fibroblasts were used to study the cell-hydrogel interactions. Both the hydrogels and their degradation products are biocompatible, as proved by the long-term cell viability assay. When cultured with cells, the hydrogel underwent a degradation within 4 weeks, with an obvious loss of cohesiveness. The good biocompatibility and biodegradability was further demonstrated in mice subdermal implantations. Lastly, in vitro and in vivo depositions of extracellular matrix in hydrogels were demonstrated by SEM analysis.
The method of preparation of cross-linked HA from an oxidized hyaluronan and gelatin by a water-in-oil-emulsion method, where a 3-dimensional hydrogel is formed in the absence of any external cross-linker, was described in the publication of Weng et al., Biomaterials 2008, 29, (31), 4149-56. In this work, incorporation of model drugs into the hydrogel structure (encapsulation) and their releasing through macrophages were studied by HPLC.
The preparation of elastic hydro gels by coupling the HA oxidized to HA-aldehyde by means of sodium periodate and the HA modified with adipic acid dihydrazide, was described by Sahiner et al., (Scheme 5), J. Biomater. Sci. Polym. Ed 2008, 19 (2), 223-43.
The resulting derivatives did not have any observable effect on the proliferation of the cultured fibroblasts, as shown by a MTT assay.